1. Technical Field
The present invention relates to a method and reagents for a fluorescence polarization immunoassay procedure for determining the presence or amount of alkaloids of the opiate class or their metabolites in fluids, especially biological fluids such as urine, serum or plasma, and to methods for making the reagents. More specifically, the invention relates to (1) reagents (tracers and antibodies) for determining the presence or amount of opiate alkaloids and/or their metabolites in a sample; (2) immunogen compounds used to raise the antibodies; (3) synthetic methods used to prepare the tracer and immunogen compounds; and (4) analytical methods for conducting the assay.
2. Background Art
Plants of the poppy family, particularly Papaver Somniferum, produce as a result of their secondary metabolism significant amounts of a number of pentacyclic alkaloids known as opiates. The most important ones are morphine ( FIG. 1) and codeine (FIG. 2). These compounds have been employed medicinally from ancient times for their narcotic, analgesic or anesthetic properties. It has also been known from antiquity that repeated use of these compounds leads to a craving for or addiction to them which tends to progress to a level where it interferes with the normal functioning of the individual so affected.
In attempts to alter their pharmacological properties, medicinal chemists have produced a large number of semi synthetic derivatives of the naturally-occurring opiates which retain the pentacyclic alkaloid skeleton, some of which are produced commercially for medicinal use. Important examples include diacetylmorphine (heroin, FIG. 3), Hydromorphone (FIG. 4), Hydrocodone (FIG. 5), Oxycodone (FIG. 6), Oxymorphone (FIG. 7) and Levorphanol (FIG. 8). These derivatives retain the addiction liability of the parent compounds to a greater or lesser degree.
Because of the problems created by abuse of the opiates, the production, distribution and use of most members of this class are rigidly controlled throughout the Western world. These efforts are supported in part by methods for detecting their unauthorized consumption which are rapid, reliable and selective for the opiates and/or their metabolites. Such testing is most frequently carried out on urine samples, since they are normally more accessible than blood samples. Other biological fluids have not been investigated extensively as media for these analyses.
In the past, opiates have been detected in biological samples by thin layer chromatography (TLC), high pressure liquid chromatography (HPLC), gas chromatography (GC), radioimmunoassay (RIA) or enzyme immunoassay (EIA). However, these assay methods are not without drawbacks. TLC is labor intensive and lacks sensitivity. Both HPLC and GC are labor intensive, requiring highly trained personnel to carry out extractions of the analyte from the biological matrix, while GC requires a derivativation step as well. RIA reagents degrade spontaneously, require burdensome methods of protecting and monitoring the safety of the personnel involved, and generate hazardous waste which must be disposed of in a secure manner. EIAs are subject to variability due to thermal and/or bacterial liability of reagents and to matrix effects which alter enzyme activity.
Fluorescence polarization immunoassay (FPIA) procedures provide a reliable quantitative means for measuring the amount of tracer antibody complex produced in a homogeneous competitive binding assay. Typically, in such a competitive binding immunoassay a ligand (a substance of biological interest to be determined by the technique) competes with a labeled reagent, or "liqand analog," or "tracer," for a limited number of receptor binding sites on antibodies specific to the liqand and ligand analog. The concentration of ligand in the sample determines the amount of ligand analog which binds to the antibody: the amount of ligand analog that will bind is inversely proportional to the concentration of ligand in the sample, because the liqand and the ligand analog each bind to the antibody in proportion to their respective concentrations.
Fluorescence polarization techniques are based on the principle that a fluorescent labeled compound, when excited by plane polarized light, will emit fluorescence having a degree of polarization inversely related to its rate of rotation. Accordingly, when a tracer-antibody complex having a fluorescent label is excited with plane polarized light, the emitted light remains highly polarized because the fluorophore is constrained from rotating between the time that light is absorbed and emitted. In contrast, when an unbound tracer is excited by plane-polarized light, its rotation is much faster than that of the corresponding tracer-antibody conjugate. As a result, the light emitted from the unbound tracer molecules is depolarized.
Such fluorescence polarization techniques have been applied in U.S. Pat. No. 4,420,568 to Wang, et al., which is directed to the use of a triazinylaminofluorescent moiety as the fluorophore.
Opiate antigen conjugates and antibodies have been described in U.S. Pat. No. 3,709,868 to Spector, in U.S. Pat. No. 3,867,366 to Rubenstein and Ullman, in U.S. Pat. No. 4,022,878 to Gross, in Science 176, 1143 (1972) and Science 178, 647 (1972) by Wainer, et al., in Japanese Journal of Pharmacology 24, 165 (1974) and op. cit. 24, 707 (1974) by Koida, et al., in Proceedings of the Society for Endocrinology 64, 6P (1975) by Morris, et al , in Research Communications in Chemical Pathology and Pharmacology 29, 535 (1980) and op. cit. 32, 545 (1981) by Dixon, et al., in Journal of Pharmaceutical Sciences 69. 160 (1980) by Pittman, et., in European Journal of Clinical Pharmacology 18, 339 (1980) by Bartlett, et al., and in Clinical Chemistry 27, 1524 (1981) by Findlay, et al. Fluorescein conjugates of opiates have been described in U.S. Pat. Nos. 3,998,943 and 4,255,329 to Ullman, in U.S. Pat. No. 3,935,074 to Rubenstein and Leute, in the Journal of Biological Chemistry 251, 4172 (1976) by Ullman et al. and in Life Sciences 33 Sup. I, 423 (1983) by Kolb et al.
The present invention offers an advance in the art beyond that described supra, particularly in that effective immunogens, selective antibodies, highly sensitive fluorescent tracers, methods for preparing the antibodies and fluorescent tracers, and an assay employing the tracers and antibodies are provided specifically for the determination of one or more opiate alkaloids in a sample. An assay conducted in accordance with the present invention is particularly accurate, as will be explained infra.